可以使用转录激活因子样效应核酸酶(TALEN)有效地修饰感兴趣的基因(Christian等人。,2010;李等人。,2011).然而,如果目标基因对发育至关重要,生长和生育,在F0青蛙中使用具有高诱变活性的TALEN可能导致发育障碍或不育,这将减少F1后代的数量,并使F1表型分析变得困难。我们使用非洲爪狼的DEADSouth基因的3'非翻译区(DS-3')来解决这个问题,因为已知向mRNA中添加DS-3'会诱导原始生殖细胞(PGC)特异性表达,并降低非洲爪的mRNA在体细胞中的稳定性。起初,我们在EGFP终止密码子的下游插入了X.tropicalisDS-3',并证实了在PGCs中特异性检测到EGFP表达3周。因此,我们在TALEN编码序列的终止密码子下游插入了DS-3'。选择酪氨酸酶基因作为TALEN的靶基因,因为该基因的双等位基因突变容易被白化表型辨别。当受精卵显微注射与DS-3融合的TALENmRNA时,他们的精子和卵母细胞的靶基因修饰率很高(84-100%),而体细胞中的核苷酸改变率较低(0-45%).
A gene of interest can be efficiently modified using transcription activator-like effector nucleases (TALENs) (Christian et al., 2010;Li et al., 2011). However, if a target gene is essential for development, growth and fertility, use of TALENs with high mutagenic activity in F0 frogs could result in developmental disorders or sterility, which would reduce the number of F1 progeny and make F1 phenotypical analysis difficult. We used the 3\' untranslated region of DEADSouth gene (DS-3\') of Xenopus tropicalis to solve this problem, because the addition of the DS-3\' to mRNA is known to induce primordial germ cell (PGC)-specific expression and reduce the stability in somatic cells of mRNA in Xenopus laevis. At first, we inserted the X. tropicalis DS-3\' downstream of the EGFP termination codon and confirmed that the EGFP expression was specifically detected in PGCs for three weeks. Therefore, we inserted the DS-3\' downstream of the termination codon of the TALEN coding sequence. The tyrosinase gene was selected as the target gene for TALEN because the bi-allelic mutation of this gene is easily discernible by the albino phenotype. When fertilized eggs were microinjected with TALEN mRNAs fused to the DS-3\', their sperm and oocytes had a high rate (84-100%) of target-gene modification in contrast to the lower rate (0-45%) of nucleotide alteration observed in somatic cells.